Validation of a metabolite–GWAS network for Populus trichocarpa family 1 UDP-glycosyltransferases

Metabolite genome-wide association studies (mGWASs) are increasingly used to discover the genetic basis of target phenotypes in plants such as Populus trichocarpa , a biofuel feedstock and model woody plant species. Despite their growing importance genetics metabolomics, few mGWASs experimentally validated. Here, we present functional genomics workflow for validating mGWAS-predicted enzyme–substrate relationships. We focus on uridine diphosphate–glycosyltransferases (UGTs), large family enzymes that catalyze sugar transfer variety secondary metabolites involved defense, signaling, lignification. Glycosylation influences physiological roles, localization within cells tissues, metabolic fates these metabolites. UGTs have substantially expanded P. presenting challenge large-scale characterization. Using high-throughput assay, produced substrate acceptance profiles 40 previously uncharacterized candidate enzymes. Assays confirmed 10 13 leaf mGWAS associations, focused metabolite screen demonstrated varying levels specificity among UGTs. A binding case study UGT-23 rationalized observed enzyme activities including glycosylation trichocarpinene produce trichocarpin, major higher-order salicylate trichocarpa. identified putatively lignan, flavonoid, salicylate, phytohormone metabolism, with potential implications cell wall biosynthesis, nitrogen uptake, biotic abiotic stress response determine sustainable biomass crop production. Our results provide new support silico analyses evidence-based guidance vivo